This invention relates to a method of increasing the solubility of flavonols and their related compounds in water and aqueous solutions, and to a method of providing soluble flavonol-containing compositions.
Polyphenols are those compounds which comprise more than one phenolic group. Among the polyphenols are the following classes: flavonoids (a term often used to denote polyphenols in general, but more commonly in Europe to denote only the flavones), the flavanols, proanthocyanidins (also called procyanidols, procyanins, procyanidins and tannins) and anthocyanins.
The flavones are compounds with a basic structure shown in FIG. 1 in which two benzene rings (A and B) are linked with a heterocyclic six member ring C containing a carbonyl group. Ring B can be joined in position 2 (as illustrated) to give a flavone or to position 3 to give an iso flavone. Hydroxylation can occur at one or more of positions 3, 5, 7, and 3xe2x80x2, 4xe2x80x2, 5xe2x80x2 to give compounds called flavonols. Typical examples of flavonols are: quercetin (hydroxylated at positions 3, 5, 7, 3xe2x80x2, 4xe2x80x2), kaempferol (hydroxylated at positions 3, 5, 7, 4xe2x80x2), and myricetin (hydroxylated at positions 3, 5, 7, 3xe2x80x2, 4xe2x80x2, 5xe2x80x2). Flavonols can exist naturally as the aglycone or as O-glycosides (e.g. with D-glucose, galactose, arabinose, rhamnose etc). Other forms of substitution such as methylation, sulphation and malonylation are also found.
The flavanols have a basic structure shown in FIG. 2. The two most common flavanols are catechin (hydroxyl groups at positions 5, 7, 3xe2x80x2, 4xe2x80x2) and its stereo-isomer epi-catechin. The hydroxyl groups can be esterified with gallic acid. The proanthocyanidins are polymers of catechin and/or epicatechin and can contain up to 8 or more repeat units. These compounds are often called proanthocyanidins, procyanidins or tannins.
The anthocyanins are colored substances, sometimes called anthocyanidins. The monomeric anthocyanins have a basic structure as shown in FIG. 3. Typical examples are: cyanidin (hydroxylated at positions 3, 5, 7, 3xe2x80x2, 4xe2x80x2), delphinidin (hydroxylated at positions 3, 5, 7, 4xe2x80x2, 5xe2x80x2) and pelargonidin (hydroxylated at positions 3, 5, 7, 3xe2x80x2). The hydroxyl groups are usually glycosylated and/or methoxylated (e.g. malvidin is substituted at the 3xe2x80x2 and 5xe2x80x2 hydroxyl groups and paeonidin and petunidin are substituted at the 3xe2x80x2 hydroxyl group). In addition polymers of these anthocyanins exist which are classified as polymeric anthocyanins.
Within the general term xe2x80x9cpolyphenolsxe2x80x9d are also included the dihydroxy and trihydroxy benzoic acids and the phytoalexins, a typical example of which is resveratrol.
Polyphenols are found in various amounts in large numbers of natural products especially plant material such as fruit and vegetables. A particular rich source is the grape, in which the polyphenols are plentiful in the skins and seeds, but not in the pulp. During the manufacture of grape juice, quantities of polyphenols are expressed into the juice, and the polyphenol content will depend on such factors as the type of grape, the climate in which it is grown, and the manufacturing process used in making the juice. Some grape juice, especially that made from the Concord grape, may contain as much as 2.5 g polyphenol per litre of juice. Grape skins and seeds are commercially extracted with water and other solvents to obtain polyphenols. In addition, polyphenols from grape skins and seeds become incorporated into wine during the vinification process. Red wine is made by maintaining contact between the fermenting liquor and the crushed grape residue (pomace) for prolonged periods, whilst in the manufacture of white wine the grape skins are removed relatively quickly. Accordingly, wine in general, and red wine in particular, contains reasonable amounts of polyphenols, amounting to about 1-3 g/L and is thus a potential commercial source of polyphenolic compounds.
Polyphenols are known to have antioxidant properties and have potential use in the food, cosmetic and pharmaceutical industries. Among the polyphenols, the flavonols have been shown to have many useful properties as antioxidants, and to decrease platelet stickiness.
Epidemiological studies have shown that countries and people with a high flavonol intake have less coronary heart disease (Hertog et al, 1995 Arch. Int. Med. 155, 381-6).
In unprocessed fruit and vegetables the flavonols occur as glycosides and the aglycone is absent. The most abundant flavonol is quercetin. Among vegetables the highest concentration of quercetin glycosides is in onions (3 to 500 mg/kg), kale (100 mg/kg) French beans (30 to 45 mg/kg), and broccoli (30 mg/kg). Among fruit examined quercetin concentration averages 15 mg/kg, with apples having the highest concentration of 21 to 72 mg/kg (Hertog et al, 1992 J. Ag. Food Chem. 40, 2379-83).
Flavonol glycosides are present in grapes and values ranging from 8 to 97 mg/Kg fresh weight have been reported (Macheix et al, 1990 Fruit Phenolics pp 378 CRC Press Boca Raton). During the fermentation process, some of the sugar is split off and the aglycone formed. On average about 50% of the flavonol exists in wine as the aglycone. The flavonol content of grape skins and wine is very variable and depends on the variety of grape and more especially the amount of sunshine in which the grapes are grown. The flavonols have a yellow color and act as filters to blue and ultraviolet light which is very injurious to the grape. During periods of intense sunlight more flavonols are synthesized to protect the grape, and consequently the grape skins and the wine from which it is made has a high concentration of flavonols (Price et al, Am. J. Enol. Vitic. 46 187-194, 1995).
Some wines in France have only 5 mg/L flavonol (calculated as aglycone) whereas up to 150 mg/L have been reported in some Californian wines. The flavonols are virtually absent from the pulp and grape seeds and only trace quantities are present in commercial anthocyanin powders extracted from pomace after making red wine.
Although grape juice is often less rich in polyphenols than wine, it contains flavonols and is a readily available commodity, and can also be used as a source of polyphenols.
Most flavonols enter the diet as glycosides with the glucosides and rutin as the most common flavonols consumed (Bokkenheuser and Winter, 1988 Prog. Clin. Biol. Res., 280 142-148). The absorption of flavonols in man has been a matter of some controversy. It was estimated that the absorption of quercetin aglycone was only 0.3 to 0.5%. Thus with an intake of 50 mg, only some 0.25 mg would be absorbed (Formica et al Fd. Chem. Toxic. 1995 33 1061-1080). However recent work in ileostomy subjects showed that the absorption of quercetin aglycone was 25% and the absorption of quercetin from onions (where it exists chiefly as the glucoside) was 52% (Hollman et al Am. J. Clin. Nutr. 1995 62, 1276-1282). More recently it has been shown that the glucoside is more rapidly absorbed than the rutinoside (Hollman et al, 1997 Crit. Reviews in Science and Nutrition 37, 719-738). One of the reasons for the poor absorption of flavonols either as the aglycone or the glycosides is that they are almost insoluble in water and lipid solvents, although sparingly soluble in ethanol. Thus, on oral administration, a very high proportion of the flavonols are excreted because they are in a physical state which does not favour absorption. Thus, whilst the flavonols are considered pharmacologically beneficial, they are poorly absorbed because they are generally of low solubility. If the flavonols could be made available in a water-soluble form they would be absorbed more easily and be biologically available following oral consumption.
The anthocyanins are water soluble pigments which are responsible for the attractive colors of many flowers, fruit and leaves. They can be easily extracted from plants by acidified alcoholic solvents and many are available commercially as food colorants. They are usually supplied with malto dextrin as a diluent in a concentration suitable for inclusion in beverages or other foods (Timberlake 1980 Food Chemistry 5, 69-80).
The anthocyanins are known to react with the flavonol glycosides in a phenomenon which is described as co-pigmentation (Scheffeldt et al 1978, J. Food Science 43, 517-520). For example, rutin was found to intensify the colour of malvidinxe2x80x943, 5, diglycoside and to shift the maximum peak of absorption. The process is believed to be the result of a hydrogen bonding mechanism.
Although the flavonols are insoluble in water, they exist in wine in quite high concentrations which vary according to the type of grape used and country of origin (McDonald et al, J. Agric. Food Chem. 1998 46, 368-375). The concentration of total flavonols (expressed as the algycone) in wine was found to range from 4.6 to 41.6 mg/L. Since wine contains about 12% ethanol, and flavonols are soluble in ethanol, it was reasonable to suppose that the relatively high solubility of flavonols in wine was due to the presence of ethanol.
It would be a great advantage to provide flavonols for oral administration in a soluble form, in water or aqueous mixtures or alternatively to increase the concentration of flavonols in ethanol containing beverages. Being in a soluble form would make possible the use of flavonols in beverages, and potentially increase their bio-availability.
In a first aspect the invention provides a method of increasing the solubility in water of a flavonol component of a flavonol-containing composition, the method comprising the steps of: providing a flavonol-containing composition; providing an anthocyanin-containing composition; and mixing the two compositions. The inventor has found that the presence of anthocyanin in a composition significantly increases the water-solubility (and hence bio-availability) of flavonols, especially at neutral or acidic pH values.
In a second aspect, the invention provides a method of providing an aqueous solution comprising dissolved flavonols at a concentration in excess of 10 mg/L (preferably over 20 mg/L, more preferably over 50 mg/L, and most preferably over 100 mg/L), the method including the steps of mixing, in any order, water, a flavonol and an anthocyanin; and forming an aqueous solution from the mixture comprising the flavonol dissolved in the solution at a concentration in excess of 10 mg/L.